Structure prediction and expression of modified rCTLA4-Ig as a blocker for B7 molecules

Aims: CTLA4-Ig (Abatacept) has been produced to suppress immune response by inhibition of T cells functions in autoimmune disease. A new drug, which is called belatacept, has recently been developed that is more efficient. The development has been occurred by two substitutions (A29Y, L104E) in the extracellular domain of CTLA4. In the present study, the bioinformatics analysis was used in order to make a new structure that has a better function in comparison with belatacept. Methods: Firstly, eight different structures were designed. Thereafter, the secondary and 3D structures, mRNA structure, docking of chimeric proteins with CD80/CD86, antigenicity and affinity of designed chimeric molecules were predicted. Based on the criteria, a new candidate molecule was selected and its gene synthesized. The gene was cloned and expressed in E. coli BL21 (DE3) successfully. The purified rCTLA4-Ig was analyzed by SDS-PAGE, western blotting and ELISA. Circular dichroism analysis (CD analysis) was used for characterization of the rCTLA4-Ig. Affinity of rCTLA4-Ig was also evaluated by the flow cytometry method. Finally, its biological activity was determined by T cell inhibition test. Results: The results showed rCTLA4-Ig and the belatacept protein have some similarities in structure and function. In addition rCTLA4-Ig was able to bind CD80/CD86 and inhibit T cell function. Conclusion: Although flow cytomery results showed that the standard protein (CTLA4-Ig), represented better affinity than rCTLA4-Ig, the recombinant protein was able to inhibit T cell proliferation as well as CTLA4-Ig.